1. Field of the Invention
The invention relates generally to photometrically analyzing a sample in a chemical detection system. More particularly, the invention is directed to an apparatus and method for uniformly illuminating a sample in a micro-channel array or detection cell of an electrophoresis system using a prism arrangement.
2. Description of the Related Art
The separation and analysis of chemical samples is widely used in both chemistry and biotechnology. In order to increase the speed and efficiency at which chemical samples are evaluated, chemical samples are separated into their component parts and simultaneously analyzed.
One such separation technology, electrophoresis, is used in DNA sequencing, protein molecular weight determination, genetic mapping, and other types of processes used to gather large amounts of analytical information about particular chemical samples. Electrophoresis is the migration of charged colloidal particles or molecules through a solution under the influence of an applied electric field usually provided by immersed electrodes, where the colloidal particles are a suspension of finely divided particles in a continuous medium.
Historically, a polymer gel containing the finely divided particles was placed between two glass plates and an electric field applied to both ends of the plates. This method, however, offered a low level of automation together with long analysis times.
More recently, capillary electrophoresis (hereinafter xe2x80x9cCExe2x80x9d) was developed, which has the added advantages of speed, versatility and low running costs. Operation of a CE system involves application of a high voltage (typically 10-30 kV) across a narrow bore capillary (typically 25-100 xcexcm). The capillary is filled with electrolyte solution which conducts current through the inside of the capillary. The ends of the capillary are dipped into reservoirs filled with the electrolyte. Electrodes made of an inert material such as platinum are also inserted into the electrolyte reservoirs to complete the electrical circuit. A small volume of sample is injected into one end of the capillary. Application of the voltage causes movement of sample ions towards their appropriate electrode. Different sample ions arrive at a detection part of the capillary at different times. The sample may be labeled with a fluorescent marker so that when the sample passes through a beam of light at the detector the fluorescent marker fluoresces and the fluorescence is detected by a detector, usually a UV detector, as an electric signal. The intensity of the electric signal depends on the amount of fluorescent marker present in the detection zone. The plot of detector response versus time is then generated, which is termed an electropherogram.
CE is a particularly preferred separation method, as it allows the use of high electric fields due to the capillary tube efficiently dissipating the resulting heat produced by the electric field. As such, the separations achieved are much better than the more traditional electrophoretic systems. In addition, multiple capillary tubes may be closely spaced together and used simultaneously to increase sample throughput.
In traditional CE systems, analysis or detection of the separated components is performed while the sample is still located within the capillary and may be accomplished using photometric techniques such as adsorbance and fluorescence. These photometric techniques direct excitation light toward the capillary tube. Light emitted from the sample (e.g., fluorescence) is then measured by a detector, thereby providing information about the separated components. Therefore, in these systems, excitation light directed at the sample, as well as light emitted from the sample, must be transmitted through the capillary""s walls. A drawback of this approach is that the fused silica capillaries typically used in capillary electrophoresis are poor optical elements and cause significant scattering of light. The problem associated with light scattering is exacerbated by having multiple capillaries disposed side-by-side, as scattered excitation light from one capillary interferes with the detection of samples in neighboring capillaries.
One approach to solving the problem of on-capillary detection has been to detect a sample after the sample emerges from the capillary in a detection cell having superior optical characteristics, e.g., a flat quartz chamber. In this system, a sample is transported from the outlet of a capillary to the detection cell by electrophoresis under the influence of the same voltage difference used to conduct the electrophoretic separation. Examples of this type of system are disclosed in U.S. Pat. No. 5,529,679, which is incorporated herein by reference.
A variation of the above system replaces the capillary tubes with a series of parallel micro-channels formed in a plate or chip, where the micro-channels are in fluid communication with a detection cell in a manner similar to that described above. This CE layout is known as a micro-channel array.
While addressing some of the abovementioned problems, the detection cell type CE system has drawbacks of its own. For example, excitation energy, such as light from a laser, has the tendency to scatter, thereby diminishing the energy""s intensity as it transmitted through the detection cell.
A partial cross-section of a prior art detection cell 102 is shown in FIG. 1A. The detection cell 102, typically made from glass substrate, forms a cavity 108, which is filled with an electrolytic polymer 110 containing a sample to be detected. The cavity 108 is then typically covered with a transparent cover 118. Excitation light 104, typically from a laser, enters the detection cell 102 at a first end 112. Because the first end 112 is normal to the excitation light 104, the light 104 does not scatter, i.e., reflect or refract, when passing into the detection cell, from air to glass. However, when the light 104 passes through the boundary 106 between the detection cell and the polymer 110, the light is refracted. This is due to the angle or slope of the boundary 106, and the difference in refractive indices of the glass and polymer. The angle or slope of the boundary 106 is caused by current etching and mastering technologies, which are typically unable to produce optically flat vertical cavity walls in glass or plastic cavities 108 of the required dimensions.
The refracted light obeys the law of refraction, i.e.,
RII sin(AI)=RIR sin(AR)
where RII=first refractive index;
AI=angle of incidence;
RIR=second refractive index; and
AR=angle of refraction.
As the polymer has a refractive index (approximately 1.41) less than the refractive index of glass (approximately 1.52), the angle of refraction is larger than the angle of incidence and the light bends further away from the normal to the boundary 106. Much of the excitation light is lost due to light escaping 116 out of the detection cell instead of being trapped in the cavity by Fresnel reflection. This degrades the intensity of excitation light incident on the samples, which in turn adversely affects the strength of the detected signal. Furthermore, refracted light rays may also reflect 114 off the internal surfaces of the cavity 108 causing interference and, therefore, degradation of the detection signal. In other words, the curved or angled interfaces or boundaries in combination with the unfavorable refractive index change at the glass to polymer boundary or interface, leads to unsatisfactory light intensity and quality, and consequently poor sample detection.
Moreover, the first end 112 through which the light first passes must be optically flat so that the light is not distorted. This requires the first end 112 to be polished, which is both expensive and time consuming.
Also, the substrate through which the light passes before entering the cavity may contain defects, such as voids, contaminants, or non-homogeneous material that creates density gradients. These defects can cause the light to scatter, refract, reflect, or the like, all of which degrade the light quality and hence detected signal.
In light of the above, there is a need for a more efficient means for directing light into a cavity while addressing the abovementioned drawbacks.
According to an embodiment there is provided a detection cell of an electrophoresis system. The detection cell includes a substrate that defines a cavity. The cavity may have a substantially planar floor and at least one wall with an opening there through. The detection cell also may include a prism disposed adjacent the opening. The prism is configured to redirect light through the opening into the cavity at an angle substantially parallel to the floor.
The prism may include a transparent exit surface disposed adjacent, and bounding, the opening and a reflector inclined at an acute angle to the transparent surface. The reflector is configured to redirect light substantially orthogonally through the transparent surface into the cavity. The prism may also include a transparent entry surface disposed substantially perpendicular to the exit surface.
In another embodiment, a shaft is bored at least partially through the substrate adjacent the opening. The shaft is inclined substantially perpendicular to the floor. The prism is then positioned within the shaft.
In an alternative embodiment, the prism includes an additional reflector disposed substantially parallel to the reflector. The additional reflector is configured to redirect light from a light source at the reflector.
Further, according to various embodiments there is provided an additional prism disposed adjacent an orifice in an additional wall of the cavity opposing the opening. The additional prism is configured to redirect light exiting through the orifice away from the cavity to avoid light scatter. The additional prism may include a transparent exit surface disposed adjacent the orifice and a reflector inclined at an acute angle to the transparent exit surface. The reflector is configured to redirect light away from the cavity at an angle substantially perpendicular to the floor.
Still further, according to various embodiments there is provided a method for illuminating a chemical sample. A chemical sample is positioned in the cavity. Light is firstly directed at a prism. The prism is disposed adjacent an opening leading into a cavity containing a chemical sample. Subsequently the light is reflected within the prism to pass through the opening and into the cavity to illuminate the chemical sample.
Various embodiments address the above described drawbacks by guiding light into a detection cell using a light guide, such as a prism. The light guide provides a controlled reflector near the entry of a cavity. The reflector of the light guide is isolated from the chemistry in the cavity by a transparent surface that may form part of the light guide itself. In an alternate embodiment an additional reflector of the light guide redirects light to the reflector of the light guide so that the light may be directed into the detection cell from any chosen orientation. The transparent surface of the light guide forms part of the light guide""s wall. The various surfaces of the light guide are made optically flat to eliminate beam reshaping and refraction issues. Also, since the transparent surface is flat, the unfavorable index of the polymer does not affect the light beam entry into the cavity.
Furthermore, cavity illumination overcomes the problems of not having a clean optical surface on the edge of the substrate by bringing the light in though a shaft somewhere within the edges of the substrate. The cross-section of the shaft can be either square, round, or other polygonally shaped form.